Toxicity, arsenic speciation and characteristics of hyphenated techniques used for arsenic determination in vegetables. A review

Arsenic (As) speciation is an interesting topic because it is well recognized that the toxicity of this metalloid ultimately depends on its chemical form. More than 300 arsenicals exist naturally. However, As can be present in four oxidation states: As−III, As0, AsIII and AsV. Long-term exposure to As from different sources, such as anthropogenic processes, or water, fauna and flora contaminated with As, has put human health at risk for decades. There are many side-effects correlated with exposure to InAs species, such as skin problems, respiratory diseases, kidney problems, cardiovascular diseases and even cancer. There are different levels and types of As in foods, particularly in vegetables. Furthermore, different chemical methods and techniques have been developed. Therefore, this review focuses on the general properties of various approaches used to identify As species in vegetation samples published worldwide. This includes various approaches (different solvents and techniques) used to extract As species from the matrix. Then, versatile chromatographic and non-chromatographic systems to separate different forms of As are reviewed. Finally, the general properties of the most common instruments used to detect As species from samples of interest are listed.


Introduction
Vegetables are recognized as crucial parts of the human diet, because they are a rich source of key elements like vitamins, minerals, dietary ber, and antioxidants.Consuming vegetables on a daily basis, in both raw and cooked states, is good for human growth and development as well as for preventing various diseases, 1 particularly when signicant global challenges are food instability, malnutrition, and food-related diseases, including diabetes, high blood pressure, cancer, and obesity.These issues have led to an increase in the demand for healthy foods, particularly vegetables.It also encourages the production and consumption of vegetables, which account for 86% of global market share. 2he accumulation of heavy metals and metalloids in plants and vegetables has recently received increased attention.Due to the anthropogenic activities , vegetables may be more exposed to environmental contamination than other food systems.When heavy metals are accumulated and taken up by plants in edible and non-edible fractions at a particular level, both animals and people may experience health issues. 3Environmental trace elements are harmful to human health.Numerous studies have demonstrated that these factors can have an impact on the environment and the quality of food.It is commonly recognized that heavy metal contamination can pollute human food supplies, particularly vegetables. 4Plants may become contaminated by metals or microbial development due to a variety of causes, such as the environment, pollution, atmosphere, soil, harvesting, and handling.Determining the quantity of certain metallic elements is crucial, since consuming large amounts of these elements is harmful.The World Health Organization advises adopting appropriate methods and standard measurements to assure the quality of plants and their products. 5s pollution is a recognized human carcinogen that affects hundreds of millions of people worldwide.Inorganic As is a prominent cause of skin, lung, bladder, liver, prostate, and kidney cancer in humans. 6Because it is introduced to the environment both naturally and via human activity, As is a common substance. 7It exists in the pedosphere, hydrosphere, biosphere, atmosphere, and water.The biogeochemical behaviour of As is governed by physical-chemical processes, including oxidation-reduction, precipitation/solubilization, and adsorption/desorption in addition to biological mechanisms, including microbiological processes. 8eavy metal exposure in vegetables can result from anthropogenic or natural processes.In contrast to anthropocentric metal concentration, naturally occurring metals are found in crusted materials, vapors, and particle matter from volcanoes and continental dust.The most signicant and frequent sources of metals in vegetables come from anthropogenic activities, such as extensive long-term pesticide and fertilizer usage, as well as linear, point, and surface metal emissions from industrial activities. 9Additionally, the accumulation of As in environmental samples results from both manmade (using As-based insecticides and herbicides, primarily monomethyl arsenic acid (MMA) and dimethyl arsenic acid (DMA)) and natural (volcanic eruptions) sources.Arsenic trioxides [As 2 O 3 ], which are used in cosmetics, reworks, electronics, glass, Cubased alloys, herbicides, fertilizers, pesticides, and seaweed fertilizers, are examples of As oxides used in industry, mining, agriculture, and other elds. 10herefore, this review article summarizes the methods used to extract As species from vegetables, then separating them using chromatographic and non-chromatographic tools, followed by determining the level of As as well as speciating the types using hyphenated techniques (Fig. 1).

Geochemistry of As
As is the 12 th most abundant metalloid in the human body and 20 th most abundant in nature.As and its derivatives are widely employed in a variety of industries, including agriculture, electronics, metallurgy, and chemical weapons, cattle, insecticides, and fertilizers, in addition to being used as a medicine. 11,12InAs compounds are produced when the element As, which is abundant in the earth's crust at 1.8 ppm by weight, mixes with oxygen, chlorine, and sulfur.The main source of As release and groundwater quality degradation in aquifer systems is rock-water interactions.As typically takes the three allotropic forms of black, yellow, and grey. 12As is an essential component of at least 568 minerals, which are found naturally in the minerals that comprise rocks. 10About 60% of these are arsenates, 20% are suldes and sulfosalts, 10% are oxides, and the remainder are native elements, metal alloys, arsenites, and arsenides.The principal As-bearing minerals that include As as an anion (arsenide), dianion (diarsenide), or as a sulfarsenide anion or anion(s) are the most signicant.These anions are bound to metals such Fe (löllingite, arsenopyrite), Co (cobaltite), and Ni (gersdorffite). 13The most important Asbearing minerals are orpiment (As 2 S 3 ), realgar (AsS), mispickel (FeAsS), löllingite (FeAs 2 ), niccolite (NiAs), cobaltite (CoAsS), tennantite (Cu 12 As 4 S 13 ), and enargite (Cu 3 AsS 4 ). 14Generally, in groundwater, high levels of naturally occurring As have been reported in aquifers-especially unconsolidated sediment aquifers throughout the world-and have been connected to several adverse health effects. 15As species A signicant part of the human diet is vegetables which can collect As in their edible and non-edible compartments by absorbing it from polluted agricultural soil and water. 16There are numerous inorganic and organic forms of As with different toxicity characteristics.As 0 (metalloid arsenic, 0 oxidation state), As III (trivalent state, e.g.arsenites), As −III (trivalent state, arsine and arsenide, −3 oxidation state) and As V (pentavalent state, e.g.arsenates) are the three most common valence states in which it can be found. 11,17A naturally occurring and widely dispersed metalloid, As can be found in soil, water, food, and the environment.As exposure from numerous human activities and sources, such as contaminated groundwater, has grown to be a major global concern.This is due to evidence that As has very hazardous potential to cause detrimental effects on human health.Human exposure to it has been connected to a wide range of diseases, and this poses a serious threat to people's health, economic security, and social standing, particularly in less developed nations. 17Even though As has a reputation for being hazardous, it is generally known that its toxicity relies greatly on the chemical state in which it exists and that the inorganic species As III and As V are considered to be more toxic than organoarsenic compounds (e.g.DMA and MMA). 18Table 1 shows some of the most common As species.

Allowable limit of As in vegetation
Many international organizations have established recommended upper limits for the amount of As that should be    present in foods.This is due to the heavy enrichment and biotransformation of As and its detrimental effects on human health.Any age or health condition can be negatively impacted by As, which is harmful to humans.Inorganic As (InAs) is the category of As that has the greatest risk of toxicity.As levels are monitored and controlled by the FDA in meals, nutritional supplements, and cosmetics.As cannot be completely prevented or removed from food, although its levels can be brought down.The EU has not yet dened a maximum limit for As in food products.However, in some states, the upper limits are set forth in national legislation.For instance, in the UK, 1 mg g −1 fresh weight of As is the statutory maximum for foods. 19The FDA is recommending a limit or "action level" for InAs in infant rice cereal of 100 mg kg −1 .The standard set by the European Commission (EC) for rice used in the manufacturing of food for babies and young children is comparable to this. 20The Scien-tic Panel on Contaminants in the Food Chain (CONTAM Panel) of the European Food Safety Authority (EFSA) adopted an opinion on As in food on 12 October 2009.In this opinion, the CONTAM Panel stated that the Joint FAO/WHO Expert Committee on Food Additives' (JECFA) provisional tolerable weekly intake (PTWI) of 15 mg kg −1 body weight is no longer appropriate because data have shown that InAs causes cancer of the lung and bladder in addition to skin cancer and that a variety of adverse effects have been reported at exposures lower than those examined by the JECFA.For skin lesions, bladder, lung, and skin cancers, the CONTAM Panel determined a range of benchmark dose lower condence limits (BMDL01) values between 0.3 and 8 mg kg −1 b.w. per day.The maximum values of InAs for non-parboiled milled rice (polished or white rice); parboiled rice and husked rice; rice waffles, rice wafers, rice crackers and rice cakes; and rice for the manufacturing of foods for infants and children were proposed by the EFSA to be 200, 250, 300, and 100 mg kg −1 , respectively, 21 China has established a legal cap of 150 mg kg −1 for InAs in rice and rice-based products. 22However, As exists at low concentrations in natural water, and in some locations, the levels of As in drinking water are over the maximum allowable concentration, which is 50 mg L −1 , whereas the recommended threshold is 10 mg L −1 , according to the Environmental Protection Agency (EPA). 23

Toxicity of As
Elemental speciation has been a well-established subject of study in recent years.5][26] As is typically thought to be metabolized mostly through methylation to lessen its toxicity and this is considered to be a detoxication process.However, by 2020, it was discovered that the synthesis of methylated metabolites containing trivalent As was necessary for at least some of the harmful consequences linked to As exposure.These results were in line with changes in the dynamic behaviour of As brought on by methylation because the trivalent oxidation state of As is linked to increased effectiveness as a cytotoxin and clastogen. 27he InAs species (As III and As V ) are categorized as species that cause cancer. 28Whereas the organic As (OAs) species (MMA and DMA), despite being less toxic than InAs, are nevertheless categorized as species that provoke cancer, 29,30 while AsC and AsB are categorized as non-toxic As species. 31However, intermediate metabolites, such as monomethylarsonous acid (MMA III ) and dimethylarsinous acid (DMA III ), are more toxic than As III , As V , DMA V or MMA V . 32

As and human health
Researchers have identied As as a signicant danger factor for both food and water.It exists both naturally and as a result of human activity in the environment in organic and inorganic forms.Exposure to As may have negative health consequences on people as well as other living things.It can also have a variety of side effects, such as skin changes, respiratory issues, cardiovascular problems, digestive system issues, genotoxicity, and mutagenic and carcinogenic effects. 16he WHO deemed this situation to be the "largest mass poisoning of a population in history" because Bangladesh as a whole experienced the worst As poisoning public health danger. 33In acute toxicity, As, a toxic metalloid, can cause nausea, vomiting, and severe diarrhea; in chronic toxicity, it can cause cardiovascular disease, diabetes, bladder cancer, and kidney cancer. 34Lung, bladder, kidney, skin, and liver malignancies, neurological disorders, cardiovascular diseases, hypertension, gangrene, diabetes, respiratory diseases, renal diseases, and reproductive diseases are among the prevalent adverse health effects of As exposure. 35The amount of ingested As, dietary status, length of exposure, and immune response of the individual are the key factors inuencing the severity of As poisoning.Skin lesions, such as arsenicosis, are the telltale signs of chronic As exposure.Arsenicosis is a worldwide issue, not just a local one in Bangladesh, with Asian nations like Bangladesh, India, and China being the worst impacted. 36More  2 effects of As exposure on the human body are illustrated in Fig. 3.

How As moves into plants and vegetables
In order to understand the mechanism supporting As transport, metabolism, and the detoxication mechanism responsible for scavenging As toxicity in diverse plants including rice, a number of genomes, proteomics, and metabolomic investigations have been conducted.Heavy metals and As can enter the body through specic routes.These routes include inhaling polluted dust, directly ingesting contaminated soil on the surface of foods, and ingesting plant-based foods that have been poisoned at the location of their growth.Vegetables have been reported to have the highest percentage of exposure to As, followed by fruit and fruit juices, rice, and other foods, in locations where there is no water contamination. 38s is easily accumulated by plants, which makes it easier for As to go from the soil into the food chain.In the oxic layer of soils, arsenate, which behaves chemically in a similar way to phosphate, is the main InAs species.As a result, As V can enter plant species via the phosphate transporter system, whereupon the As V in plant biomass may be changed into As III .The amount of phosphate in the soil affects the absorption and phytotoxicity of As.As can displace phosphate in soil particles at low concentrations, increasing absorption and phytotoxicity, whereas high concentrations of phosphate compete with As at root surfaces, decreasing uptake and phytotoxicity.18,39 In addition, As behaviour in the soil environment is inuenced by microbial activities and hence As availability in the soil-plant system.32 7 As speciation in vegetables

Common extraction techniques and solvents
For information on accurate As speciation, it is necessary to maintain the concentration and chemical composition of the original species during the sample preparation and extraction procedures. 40The preferred extraction method for a given application reects both the matrix and the target species.For the precise measurement of As species in vegetables and plants using HPLC-ICP-MS, a gentle and effective extraction procedure is needed. 18For the detection of As in vegetables, advanced techniques, such as shaking, sonication, accelerated solvent extraction and microwave-assisted extraction (MAE), have also been used (Table 2).However, for many matrices, MAE is an effective substitute for traditional methods because it provides acceptable and repeatable efficiency, shorter extraction duration, use of less solvent, and the ability to do numerous extractions quickly.Numerous investigations on the speciation of As have used this method.Due to the small number of variables involved, such as solvent selection, solvent volume, temperature, extraction time, power, and matrix characteristics, optimization is simple. 41,42ue to their ability to selectively hydrolyze the important parts of the cell, enzymes may be used as an extraction tool for As species.As a result, the amount of material needed could be greatly reduced, necessitating less sample dilution and enabling the analysis of As species that cannot be extracted using standard methods (water or water/methanol). 43Enzymes have also been employed to treat some food items; freeze-dried apple samples were treated with amylase.Amylase degrades the cellulose in freeze-dried apple samples, increasing the yields of As species during extraction.Acetonitrile-water extraction may then be used to complete the process. 43,44However, the  extraction efficiency for As utilizing cellulase as the extraction agent was shown by cellulose to vary signicantly for GBW10015 materials.For GBW10015-spinach, cellulase had an extraction efficiency of 119%.Additionally, some As species were retained in the residue because cellulase was unable to completely remove all the As from GBW10015-spinach.Because of this, cellulase is not a suitable extraction agent for extracting As species from plants and vegetables. 18There are many different extraction techniques that have been used for full, total inorganic, and total As speciation.The most commonly used extraction agents are water, methanol, methanol-water solvent systems, and occasionally, though rarely, acetonitrile-water solvent systems.Sequential extractions are also popular.
Although water is an inexpensive and affordable solvent that can be used to remove As species in a plant and vegetable matrix, it is incapable of extracting InAs species (As III and As V ) that bind protein. 457][48] Triuoroacetic acid can increase the effectiveness of extraction, but it also has the potential to change the species of As. 49 Although phosphate in the plasma could damage the cones of the mass spectrometer due to polymeric deposition and chlorine could interfere with the detection of 75 As + by forming the polyatomic species 40 Ar 35 Cl + in the subsequent ICP-MS analysis, diluted inorganic acid solutions (such as HNO 3 , H 3 PO 4 , and HCl) work well at extracting As species from plant tissues. 50,511 There is a need to separate these arsenical efficiently in real samples.

Capillary electrophoresis (CE).
Due to its excellent separation efficiency and relatively gentle separation conditions, which would aid in conserving the original As species in samples, CE offers an alternative separation method to HPLC.Because CE is a separation method that operates at the nL min −1 level and because its (electro-osmosis dewatering) EOF is substantially lower than the uptake rate for traditional micro-concentric nebulizers of ICP-MS, coupling of CE and ICP-MS presents a difficult design challenge. 62Since CE has several distinct advantages over GC or HPLC methods, including high resolving power, quick and effective separation, minimal reagent consumption, and the potential for separation with only minor disruptions of the existing equilibrium between different species, CE is an appealing technique for elemental speciation. 63Separation of different organic and inorganic As species has been identied using CE.
7.2.1.2Chromatographic methods 7.2.1.2.1 Ion chromatography.Cation-exchange chromatography is utilized with positively charged target molecules.Because the pH for chromatography is lower than the isoelectric point (pI), the molecule is positively charged.The stationary phase in this type of chromatography is negatively charged, and positively charged molecules are loaded to be dragged to it. 64ased on their physicochemical characteristics (pK a values), As V , MMA, and DMA are deprotonated to create anionic species under neutral pH, whereas As III exists as a neutral species.Due to variations in their anionic nature, anion-exchange chromatography is a potential alternative for separating these prevalent As species.Due to its low pK a1 (2.19) and pK a2 (6.98) values, As V has the strongest negative charge in most mobile phases and elutes slowly.Given that As III has high pK a values (pK a1 = 9.23, pK a2 = 12.13, and pK a3 = 13.4), it oen occurs as a neutral molecule, which results in relatively minimal retention.Many As compounds, including As III , As V , MMA, DMA, AsB, AsC, oxoarsenosugars (oxoAsS), thio-arsenosugars (thioAsS), and phenylarsenicals, have been analysed using anion exchange chromatography.Strong anion-exchange columns, such as PRP-X100, are the most popular type of column used for As speciation studies. 65hen the stationary phase is positively charged and negatively charged molecules are loaded to be attracted to it (i.e., the pH for chromatography is larger than the pI), this process is known as anion-exchange chromatography.For the speciation investigation of positively charged As molecules, such AsB, AsC, TMAO, or TMA, cation-exchange chromatography is frequently utilized.Similar to anion exchange, cation-exchange chromatography operates by interacting with cationic analytes through the use of a negatively charged stationary phase.Stronger positively-charged analytes retain more information, which is directly correlated with the retention of arsenicals.In 15 minutes, Wolle et al. separated As III , MMA, As V , DMA, AsB, and TMAO using a strong cation-exchange (PRP-X200) column, with AsC and TMA co-eluting. 66

Ion-pair chromatography (IPC). IPC has commonly been used for
As speciation studies because it can distinguish between ionic and neutral species.Ion-pair reagents are used in the mobile phase of a typical reversed-phase column (C18) in the IPC process.While the hydrophobic area of the ion-pair reagent interacts with the stationary phase, the charged group of the reagent interacts with the analyte.Tetraalkylammonium, tetrabutylammonium, and tetraethylammonium are frequently utilized as ion-pair reagents for the speciation of anionic and neutral As species.The ion-pair separation of cationic and neutral As species frequently use alkyl sulfonates, such as hexane sulfonic acid and 1-pentane sulfonic acid.The two organic modiers that are used most frequently are acetonitrile and methanol.Usually included in the mobile phase, they reduce retention duration and alter selectivity.The charged As species must pass through the ion-pair reagents and the hydrophobic stationary phase in order to interact with the conventional stationary phase, whereas the neutral As species can do so directly.In less than 12 minutes, Morita et al. developed a mixed ion-pair method using sodium butanesulfonate and tetramethylammonium hydroxide as the ion-pairing reagents to separate As V , As III , MMA, DMA, AsB, TMAO, TMA, and AsC. 67Eight As species, As III , As V , MMA, DMA, TMAO, tetramethylarsonium, AsC and AsB, have been identied in an extract of tree moss by ion-pair reversed phase HPLC. 682.1.2.3 Reversed-phase-liquid chromatography.
Arsenolipids, which comprise fatty acids, phospholipids, phosphatidylcholines, fatty alcohols, and phosphatidylethanolamines, are particularly well analyzed by reversed-phase-liquid chromatography.Arsenolipids can be separated according to the number of carbons, the amount of double bonds, and other functional groups using standard C18 or C8 columns.Reversedphase HPLC has been used with ICP-MS and ESI-MS to measure and identify several arsenolipids in sh and algae. 69,70As III , AsB, DMA, and an arsenosugar (oxo-arsenosugar-glycerol, As 328) in extracts of commercial kelp and bladderwrack have been separated and investigated using ion-pair-reversed-phase highperformance liquid chromatography (IP-RP-HPLC) coupled to particle beam-electron ionization mass spectrometry (PB-EIMS). 71The stability of three arsenolipids-As fatty acids AsFA-362 and AsFA-388, as well as As hydrocarbon AsHC-332common components of algae-was examined in relation to sample storage and transport as well as their preparation for quantitative analyses. 702.1.2.4 Hydrophilic interaction liquid chromatography (HILIC).Typically, anion-exchange columns and non-volatile solvents, for instance with phosphate buffers as mobile phase, are used to separate highly polar As compounds.Consequently, ESI-MS compatibility is only mild.So, a technique was created using hydrophilic interaction liquid chromatography (HILIC) for the separation of the main transformation products.Thus, compatibility of the used solvents for speciation analysis with both ICP-MS and ESI-MS was accomplished.In order to determine the quantity of the produced products and characterize the primary products developed, quantication of Ascontaining species was also carried out using high-resolution electrospray (EC)-HILIC-ICP-MS.Thirteen arsenicals were detected and separated using EC-HILIC-ICP-MS in research into roxarsone electrochemical transformation products.72 Xie et al. successfully used a zwitterionic HILIC column and ICP-MS/ESI-MS to separate nine organoarsenicals (i.e., 3-nitro-4hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p-arsanilic acid (p-ASA), phenylarsine oxide (PAO), DMA, MMA, AsB, AsC and TMAO) within 45 min.73 Even though HILIC has signicant potential for separating more As species in a single run, there are not many instances of it being used for As speciation.

Size exclusion chromatography (SEC). Size exclusion chromatography is not effective for speciation investigation of tiny As compounds because the size differences between many
As species are tiny and they cannot be separated on an SEC column.The investigation of As interactions with big compounds, however, is particularly effective when using SEC.For instance, SEC is frequently used in an As-protein binding study to distinguish protein-bound from free As. 74,75With a focus on maintaining the intact proteins and their As bindings, a novel approach based on size exclusion chromatography linked to electrospray ionization mass spectrometry (SEC-ESI-MS) was developed in a study.Using SEC-ESI-MS, the simultaneous binding of phenylarsine oxide to ve distinct peptides and proteins (glutathione, oxytocin, aprotinin, lactalbumin, and thioredoxin) were examined. 76SEC was also utilized to quantify As-biomolecule complexes in Mus musculus liver extracts. 77In order to separate and collect protein-bound As from free As, SEC was utilized; then, the protein-bound As was treated with hydrogen peroxide to liberate it. 78A combination of SEC coupled with ICP-MS, as well as three-dimensional excitation-emission matrix uorescence spectroscopy combined with parallel factor analysis was used to investigate the roles of dissolved organic matter on As mobilisation and speciation in environmental water. 79

Detection of As species
As can theoretically be found using any spectrometric detector that can identify an element specically.Atomic absorption spectrometry (AAS), atomic uorescence spectrometry (AFS), and mass spectrometry (MS) are the methods that are used most frequently. 80.3.1 Atomic absorption spectroscopy.An excitation source is necessary in atomic spectrometry in order to atomize or ionize the target analyte.These methods have the benet of having innately sensitive and element-specic detection.In comparison to ame AAS, graphite furnace (GF)-AAS has been preferred for As research due to its 10-100-fold higher sensitivity.There have been reports of detection limits in the region of a few nanograms for both fraction collection and on-line coupling of HPLC with GFAAS. 11,81The baseline noise level was reduced by using a high-intensity boosted discharge hollow-cathode lamp, resulting in a lower LOD of 0.26 ppb for a sample volume of 16 L, or 4.2 pg of As. 82 The AAS with vapour generation assembly (AAS-VGA) method is well known for As trace analysis.The conversion of As V to As III is necessary for appropriate analysis of the total As mixture (As III + As V ).The free As atoms that result from the conversion of As III to AsH 3 vapor and then free As are what provide the AAS absorption signal.This is accomplished using the AAS-attached vapor generation assembly, which contains a reduction channel lled with sodium borohydride and an acid channel lled with 10 M HCl. 83ue to its usefulness, simplicity, and affordability, AAS is an extensively used technique for metal measurement.However, sample pretreatment is frequently performed before the actual detection stage in order to enhance the metrological features of AAS, particularly the sensitivity and the detection limit. 84In reality, optical spectroscopy is a useful tool for identifying As III , As V , DMA, MMA, AsC, AsB and TAMO as well as for detecting considerable hydride arsenosugars and thioarsenate production when used in conjunction with various separation methods and chemical modiers. 85,86.3.2Atomic uorescence spectroscopy.The use of HPLC in conjunction with atomic uorescence spectrometry (AFS) for As speciation is now well established and effective.AFS is a good substitute for other atomic spectrometers that are frequently used in speciation research, like AAS and ICP-MS. 87Regarding performance factors like detection limits, reproducibility, repeatability, and sensitivity for As, AFS can compete with ICP-MS.Additionally, AFS provides reduced purchase and operating costs, quicker analysis warm-up times, and simple handling.][90] The drawbacks of the conventional HG borohydride/acid system have long been the subject of research into new vapor generation systems, such as electrochemical vapor generation (ECVG) and photochemical vapor generation (photo-CVG).The selective and quantitative conversion of As III to AsH 3 on the GSH-modied graphite electrode at an applied current of 0.4 A, while both As III and As V on the Cys-modied graphite electrode could generate AsH 3 at an applied current of 0.6 A. This allowed for the speciation of As by coupling with AFS.By mixing 15 mg L −1 FeCl 3 with acetic acid and formic acid, the ultraviolet vapor generation (UVG) of As has been increased around 10fold. 91Additionally, under UV light, As III and As V could be transformed to volatile As species using a nano-Au/nano-TiO 2 composite. 92AsH 3 was produced at 0.6 A of applied current, but MMA and DMA could not form any or only a small amount of hydride under these conditions, achieving the speciation of As by coupling with AFS.HPLC-HG-AFS was employed to measure As III , As V , DMA and MMA in the roots, stems and fruits of strawberry plants. 93.3.3Atomic emission spectroscopy (AES).An alternative method for As speciation is atomic emission spectroscopy or optical emission spectroscopy (OES).Despite the ability of commercial ICP-OES to simultaneously determine many elements, the comparatively poor sensitivity can be a weakness in the study of As.On the other hand, OES is simple to miniaturize in order to create methods or devices for As analysis that can be used in the eld. 94When very low limits of detection are not required, HPLC-ICP-AES is a reliable technology for As speciation.It has been concluded that detection limits better than 10 mg L −1 are needed for As III , DMA, and 20 g L −1 for As V .The method can also be combined with HG.However, it is important to keep in mind that not all As species can be iden-tied in this way. 95HPLC-ICP-OES has been utilized to identify As species, including As III and As V , in Chinese brake ferns with limits of detection of 0.032 mg L −1 As III and 0.035 mg L −1 As V per gram of dried fern material. 96.3.4Inductively coupled plasma mass spectrometry (ICP-MS).The majority of laboratories now use this technique for As speciation.Double focusing sector eld ICP-MS allows for the immediate determination of desired elements without the need for pre-concentration or isolation.Double focusing ICP-MS offers great sensitivity over a broad linear range, low LODs, and the capability of multielement analysis. 97ICP-MS is the most widely used method for the detection of various As species due to its excellent sensitivity, selectivity, and wide dynamic range.Several strategies have been devised to minimize or diminish isobaric interference (identical mass isotopes of distinct elements present in the same sample) in the detection of As at a mass-to-charge ratio of 75. 98ICP-MS outperforms other techniques primarily due to its low detection limit range, 1-10 pg mL −1 for quadrupole instruments, broad linear dynamic range, speed, multi-element capabilities for many elements, and potential to apply isotopic studies (although not for As).ICP-MS for As speciation has certain limitations despite all these benets.Without some type of previous separation, most frequently by HPLC, the use of ICP-MS does not provide direct molecular information and it is unable to identify specic As species. 99.3.4.1 Interference can be a problem in ICP-MS.When there is an isobaric overlap caused by polyatomic ions generated by the combination of two or more atoms, interference can be a problem in ICP-MS. As 35 Cl is a signicant polyatomic interference species for As [As is monoisotopic m/z 75].Refractory oxides may occur as a result of incomplete dissociation or recombination in colder plasma areas, particularly in the boundary layer surrounding the sampler cone. 100When polyatomic interference occurs, a collision/reaction cell can be used to reduce interfering ions by introducing a collision gas (such as helium), a reaction gas (such as oxygen, hydrogen, or CH 3 F), or a combination of two gases into the ICP-MS.ICP with triple quadrupole tandem mass spectrometry (ICP-QQQ) helps to eliminate isobaric interference, lessen background noise, and enhance selectivity compared to ordinary single quadrupole ICP-MS. 98here are numerous ways to reduce this interference issue in ICP-MS.Polyatomic interference can be reduced by introducing a different gas to the argon plasma, such as nitrogen, oxygen, air, helium, or hydrogen, which can also reduce the inherent polyatomic interference. 101Due to an increase in signal and a decrease in argon and O-based interference, adding nitrogen gas to an argon plasma has been proven to be quite successful. 102However, a more contemporary method that makes use of collision cell technology is currently offered on commercial instruments for the elimination of interference.][105] Due to its sensitivity and capacity to resolve isobaric overlap, sector eld (SF)-ICP-MS is possibly the most suitable option for elemental speciation research. 106As speciation in cucumber xylem sap is one instance of an As speciation study employing this method. 107.3.5 X-ray spectroscopic techniques.X-ray spectroscopic approaches are applied for As speciation analysis in As-rich biological samples with the least amount of sample preparation. 108Numerous X-ray spectroscopic techniques have been employed to measure total As and As speciation in various solid environmental and biological samples.This work looked into the feasibility of conducting speciation analysis on solid        environmental samples without the need to extract the elemental species.As speciation has been investigated in plant material using X-ray absorption near-edge structure (XANES) 109,110 and synchrotron radiation extended X-ray absorption ne structure (EXAFS). 111.3.6Chemiluminescence (CL).CL is the term for when a chemical reaction produces light.The CL created when As and ozone (O 3 ) combine has been known for more than 30 years.In addition, the CL spectra of ozone and arsine gas are dissimilar.As is converted to arsine gas during CL analysis via hydride production, and the arsine gas is then sent to an ozone chamber via carrier gas ow for further reaction.With this method, As concentrations as low as 1 ppb can be detected.However, it is not particularly useful in the eld because a carrier gas tank is required. 112L detection is an optical detection method that provides great selectivity but at a price that is signicantly less than that of atomic spectrometric methods.It has frequently been utilized in FI systems to identify both organic and inorganic species.Using luminol-based chemiluminescence detection or chemiluminescence produced by the redox reaction of As III with permanganate in the presence of sodium hexametaphosphate, inorganic As species have both been successfully identied in FI systems.The relative detection limits for these techniques were 8 mg L −1 , 100 mg L −1 , and 0.3 mgL −1 , respectively. 113

As speciation in plants and vegetables
It has long been understood that As is a phytotoxic substance that perturbs the physiological and biochemical processes of plants.The toxicity depends on As speciation, typically, inorganic species are more poisonous to living things, such as plants, people, and other animals, than organic forms. 23Vegetables can absorb As from their surroundings (such as soil, irrigation water, and accumulated dust), leading to their future contamination. 114Therefore, for a population that consumes a lot of vegetables in As-contaminated areas, dietary intake may represent a signicant As exposure pathway.As levels in a plant's edible compartments are inuenced by the amount of As present in the soil, as well as the plant's capacity for accumulation and translocation. 115,116As is easily accumulated by vegetables, which makes it easier for As to go from the soil to the food chain.In the oxic layer of soils, As V , which behaves chemically in a similar way to phosphate, is the main InAs species.As a result, As V can enter plant species via the phosphate transporter system, whereupon the As V in the plant biomass may be changed into As III .The amount of phosphate in the soil affects the absorption and phytotoxicity of As.As can displace phosphate in soil particles at low concentrations, increasing absorption and phytotoxicity, whereas high concentrations of phosphate compete with As at root surfaces, decreasing uptake and phytotoxicity. 40Beside this, a protein transporter supports plants to absorb InAs.Usually, As compartments in roots then transfers to stem and grains. 117able 3 shows the literature on As species measured in vegetables and plants using different coupling instruments worldwide.

Certified reference materials (CRM)
There has been an increased demand for various CRM types in chemical analysis during the past few years, as well as new CRM publications regarding their advances and certication.The increase in ISO/IEC 17025 accreditation serves as an example of the rising demand for traceable and trustworthy results in analytical chemistry.The usage of CRMs is highlighted among Review RSC Advances the several technological requirements in this quality system because of its application in many processes, including method validation, prociency testing, uncertainty estimation, and quality control. 126nvironmental control laboratories should utilize CRMs to test or verify the performance of their analytical methods in order to give accurate information and to comply with the data quality objectives of the legislation. 127As-containing CRMs have been produced, but the majority of them have total-element concentration certication.Because of the growing usage of species-specic measurement, species-specic CRM materials are now essential. 11Some certied materials used for As speciation in plants and vegetables are listed in Table 4.

Conclusions
Millions of people worldwide rely heavily on edible plants and vegetables for food, yet when contaminated with toxins like As, they also pose the biggest threat to human health.The outcome of toxic As species present in vegetation on human health is of serious concern because several diseases are linked with toxic forms, especially if they surpass the allowable range.Although a certain limit for As in drinking water has been set, for vegetation this value has not yet been documented.Both can accumulate variable quantities of heavy metals as well as As species from environments such as water and soil, and anthropogenic processes.This review highlights an overview of available methods for measuring As species in edible plants and vegetables.Different chromatographic and non-chromatographic methods, and spectroscopic and coupled techniques were explained to identify and detect As species in edible plants and vegetables.The most promising and realistic methods of determining As species are recently coupled or hyphenated analytical techniques, including ICP-MS, LC-MS, and HPLC/ ICP-MS.

Fig. 1
Fig. 1 Different methods used to extract, separate and detect As species in vegetables.

Table 1
Some As compounds of environmental interest

Table 2
Summary of the most commonly used extraction procedures for extraction of As in plants and vegetables a a HPLC: high performance liquid chromatography; ICP-MS: inductively coupled plasma mass spectrometry; HG-AFS: hydride generation atomic uorescence spectrometry; IC: ion chromatography; LC: liquid chromatography.

Table 2
More than 300 As species have been identied using the most sophisticated analytical techniques.
provides a summary of the main extraction media and extraction techniques utilized to remove As species from vegetables.It should be noted that several of these procedures have fairly low extraction efficiency and are typically time-consuming.7.2 Techniques used for separation and measuring As species 7.2.1 Methods used for separation of arsenicals.

Table 3
Commonly utilized coupling techniques for detecting As species in various vegetables and plants in the literature a

Table 4
Some certified reference materials used to check validity of measuring total As and As species in plants and vegetables © 2023 The Author(s).Published by the Royal Society of Chemistry RSC Adv., 2023, 13, 30959-30977 | 30971